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      The presence of amorpha-4, 11-diene synthase, a key enzyme in artemisinin production in ten Artemisia species

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          Abstract

          Background and the purpose of the study

          Artemisinin is one of the most effective medicine against malaria, which is produced naturally by Artemisia annua in low yield. It is produced in a metabolic pathway, in which several genes and gene products are involved. One of the key genes in this pathway is am1, which encodes amorpha-4, 11-diene synthase (ADS), a key enzyme in artemisinin biosynthesis pathway. The aim of this study was to determine the presence of this gene in ten Artemisia species in order to increase the yield of production of Artemisinin.

          Methods

          The experiments were carried out using PCR. Specific primers were designed based on the published am1 gene sequence obtained from A. annua (NCBI, accession number AF327527).

          Results

          The amplification of this gene by the specific primers was considered as a positive sign for the potentiality of artemisinin production. Since the entire am1 gene was not amplified in any of the 10 species used, four parts of the gene, essential in ADS enzyme function, corresponding to a) pair site of Arg10-Pro12 in the first 100 amino acids, b) aspartate rich motif (DDXXD), c) active site final lid and d) active site including farnesyl diphosphate (FDP) ionization sites and catalytic site in the ADS enzyme, were investigated.

          Major conclusion

          The sequence corresponding to ADS active site was amplified only in A. annua, A. aucheri and A. chamaemelifolia. The negative results obtained with other species could be due to some sequence alteration, such as point mutations or INDELs. We propose A. aucheri and A. chamaemelifolia as two potential candidate species for further characterization, breeding and transferring am1 gene for artemisinin overproduction.

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          Most cited references18

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          Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L.

          In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.
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            Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin.

            The sesquiterpenoid artemisinin, isolated these from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight.
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              Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation.

              An Agrobacterium tumefaciens-mediated transformation system was developed for Artemisia annua L. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS placed under a CaMV 35S promoter) was transferred into A. annua via A. tumefaciens strain LB4404. Leaf or leaf discs were used as explants to be infected with A. tumefaciens and an optimal concentration of 20 mg/l kanamycin was applied to select kanamycin resistant shoots. Forty-five lines of resistance kanamycin shoots transformed with FDS were established. Analysis of PCR showed that at least 20 shoots transformed with the FDS gene were PCR positive. Southern blot analysis suggested the foreign FDS gene had been integrated into the A. annua genome, and Northern blot analysis revealed that the foreign FDS gene expressed at the transcriptional level in five shoot lines (F-1, F-4, F-61, F-62 and F-73 shoot lines). Analysis of artemisinin demonstrated that about 8 approximately 10 mg/g DW of artemisinin were then detected in transgenic plants regenerated from five shoot lines, this is about 2-3 times higher than that in the control.
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                Author and article information

                Journal
                Daru
                DARU
                DARU : Journal of Faculty of Pharmacy, Tehran University of Medical Sciences
                Tehran University of Medical Sciences
                1560-8115
                2008-2231
                2011
                : 19
                : 5
                : 332-337
                Affiliations
                Agricultural Biotechnology Department, Imam Khomeini International University, Qazvin, Iran
                Author notes
                Article
                DARU-19-332
                3304396
                22615678
                932aea85-4c0f-4e51-acaf-652029195137
                © 2011 Tehran University of Medical Sciences

                This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.

                History
                : 16 June 2011
                : 21 August 2011
                : 22 October 2011
                Categories
                Original Article

                Pharmacology & Pharmaceutical medicine
                asteraceae,artemisia (sweet wormwood),artemisinin,amorpha-4,11-diene synthase,pcr

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